(Title: Rational Design of pH-Low Insertion Peptides for Cargo Delivery, PI: An) Background. Cytoplasmic cargo delivery has far-reaching implications in biomedical research and medicine (e.g. delivery of probes, drugs, toxins). If the delivery vehicle can be switched on and off using a physiological input, targeted or selective cargo delivery becomes possible. In this grant, we propose to study the pH-low insertion peptide (pHLIP) as a carrier capable of transmembrane (TM) cargo delivery in response to slight acidity found in diseased tissues (e.g. cancerous tumors, ischemic myocardium). The 36-residue pHLIP peptide can insert into membrane and form a TM ?-helix in response to mild acidity. It has shown great potential for cancer diagnosis and treatment. However, its biomedical applications have been hindered by the gap between the insertion pH (pHins) of wild type pHLIP and the extracellular acidity of diseased tissues. To close this gap, appropriate tuning of pHLIP structure is necessary (i.e. SAR studies). But pHLIP is challenging to optimize because its protonation-driven insertion mechanism is not well understood. Our recent ssNMR data showed that pHLIP insertion proceeds through a defined sequence of key protonation events, which provides the necessary mechanistic insights for guiding rational design of next generation pHLIP variants. Objectives. (1) To develop new pHLIP variants with higher pHins for enhanced cargo delivery; (2) To understand their mechanism of cargo delivery in cells; (3) To study the effect of cargo on pHLIP behavior. Methods. First, we will develop a series of new pHLIP variants by replacing Asp / Glu residues involved in peptide insertion with Glu / Aad (?-aminoadipic acid) residues in a coordinated fashion, according to the ssNMR defined protonation sequence. Their membrane insertion behaviors will be evaluated using CD and fluorescence assays in lipid vesicles to select those with high pHins. Then turn-on fluorescence probes of the selected variants will be prepared and tested in cells to check if high-pHins variants interact with cells at higher extracellular pH than WT pHLIP. Next, in order to show that high-pHins variants can improve the efficiency of cytoplasmic cargo delivery (under mild acidity found in diseased states, i.e. pH 6.5-7.0), the cancer drug doxorubicin (Dox), serving as a model cargo, will be conjugated to the variants. These pHLIP-Dox conjugates will be evaluated in model membranes (CD, fluorescence biophysical assays), by imaging studies in cells, and in antiproliferation assays. Impact. The proposed work is a well-structured interdisciplinary program, which will expose students to peptide synthesis, bioconjugation chemistry, biophysical characterizations in lipid vesicles, and evaluations in cell culture. When studies proposed are completed, we will develop pHLIP variants with enhanced cargo delivery capability and reach a clear understanding about how they do so in cells. In the future, such high-pHins pHLIP variants will be used in targeted drug delivery triggered by tumor acidosis and other applications.